Project Summary In this application we propose to develop a native contrast optical spectroscopic sensing approach that identifies and characterizes subcellular structures and quantifies their properties when cells undergo pre- cancerous alterations, by using light scattering spectra as native optical biomarkers. Such a technique would enable simultaneous labeling of large number of subcellular and subnuclear structures without the use of stains and would be of great value for studying early cancer progression. The absence of stains also makes such methods easy to implement in time-course cancer progression studies and would be amenable for in vivo observations in humans. Although cellular alterations in organelle and nuclear structure are readily observed and studied in cancer, there are fundamental limitations in existing imaging techniques that prevent the study of very early stage pre-cancerous alterations. In contrast to dysplastic cellular alterations such as nuclear enlargement and organization, the earliest stages of carcinogenesis have much more subtle alterations that are not easily discernible with standard microscopy techniques. Perhaps the most often used imaging tool for observing cellular structure is fluorescence microscopy. It can achieve targeted contrast for specific organelles or proteins, however imaging in live cells remains limited to just a few types of fluorophores and therefore structure types. Although recently developed optogenetic methods and new live cell fluorescent probes have significantly improved the utility of fluorescence in living systems, the method is inherently limited to observing a few types of structures at relatively short time scales. An even more substantial limitation of conventional optical imaging is that it is subject to the diffraction limit and cannot discern the properties of cellular structures that are significantly smaller than a wavelength. On the other hand, electron microscopy imaging methods are destructive, involve extensive manipulations with the sample, and cannot be utilized in living systems. In order to overcome the limitations of both methods, a technique that is based on an entirely different physical principle is required. This method should ideally identify all important cellular structures in live cells, while simultaneously dynamically quantifying their properties when cells undergo pre-cancerous alterations.